Blood coagulation is a process consisting of a complex interaction of various blood components, or factors, which eventually gives rise to a fibrin clot. Generally, the blood components which participate in what has been referred to as the coagulation "cascade" are proenzymes or zymogens, enzymatically inactive proteins, which are converted to proteolytic enzymes by the action of an activator, itself an activated clotting factor. Coagulation factors that have undergone such a conversion and generally referred to as "active factors", and are designated by the addition of the letter "a" to the name of the coagulation factor (e.g. factor VIIa).
Activated factor X ("Xa") is required to convert prothrombin to thrombin, which then converts fibrinogen to fibrin as a final stage in forming a fibrin clot. There are two systems, or pathways that promote the activation of factor X. The "intrinsic pathway" refers to those reactions that lead to thrombin formation through utilisation of factors present only in plasma. A series of protease-mediated activations ultimately generates factor IXa, which, in conjunction with factor VIIIa, cleaves factor X into Xa. An identical proteolysis is effected by factor VIIa and its co-factor, tissue factor, in the "extrinsic pathway" of blood coagulation. Tissue factor is a membrane bound protein and does not normally circulate in plasma. Upon vessel wall injury, however, it is exposed and forms a complex with factor VIIa to catalyse factor X activation or factor IX activation in the presence of Ca.sup.++ and phospholipid (Nemerson and Gentry, Biochem. 25:4020-4033 (1986)). While the relative importance of the two coagulation pathways in hemostasis is unclear, in recent years factor VII and tissue factor have been found to play a pivotal role in the initiation and regulation of blood coagulation.
Factor VII is a trace plasma glycoprotein that circulates in blood as a single-chain zymogen. The zymogen is catalytically inactive (Williams et al., J. Biol. Chem. 264:7536-7543 (1989); Rao et al., Proc. Natl. Acad. Sci. USA. 85:6687-6691 (1988)). Single-chain factor VII may be converted to two-chain factor VIIa by factor Xa, factor XIIa, factor IXa, factor VIIa or thrombin in vitro. Factor Xa is believed to be the major physiological activator of factor VII. Like several other plasma proteins involved in hemostasis, factor VII is dependent on Vitamin K for its activity, which is required for the gamma-carboxylation of multiple glutamic acid residues that are clustered in the amino terminus of the protein. These gamma-carboxylated glutamic acids are required for the metal-associated interaction of factor VII with phospholipids.
The conversion of zymogen factor VII into the activated two-chain molecule occurs by cleavage of an internal Arg.sub.152 -IIe.sub.153 peptide bond (Hagen et al., Proc. Natl. Acad. Sci. USA 83: 2412-2416 (1986); Thim et al., Biochem. 27:7785-7793 (1988) both of which are incorporated herein by references). In the presence of tissue factor, phospholipids and calcium ions, the two-chain factor VIIa rapidly activates factor X or factor IX by limited proteolysis.
Divalent cations profoundly affect factor VIIa activity. Factor VIIa requires Ca.sup.2+ -ions and tissue factor for optimal activity. Apparently Ca.sup.2+ is required for binding to tissue factor as well as for induction of an active conformation of the FVIIa molecule. Zn.sup.2+ -ions have been shown to inhibit FVIIa activity, (Pedersen, et al.,: Thromb. Haemostas. 65:528-534 (1991); the normal concentration of free Zn.sup.2+ -ions in the blood (15 .mu.M) being 4-10 fold lower than the apparent Ki for zinc inhibition. It was suggested by these investigations that a histidine residue at position 241 immediately adjacent to the active site might be involved in coordination of the inhibitory Zn.sup.2+ -ion.
It is often desirable to selectively block or inhibit the coagulation cascade in a patient. Anticoagulants such as heparin, coumarin, derivatives of coumarin, indandione derivatives, thrombin inhibitors, factor Xa inhibitors, modified factor VII or other agents may be used.
Inhibition of coagulation is beneficial in a number of diseased states, for example during kidney dialysis, or to treat deep vein thrombosis, disseminated intravascular coagulation (DIC), and a host of other medical disorders. For example, heparin treatment or extracorporeal treatment with citrate ion (U.S. Pat. No. 4,500,309) may be used in dialysis to prevent coagulation during the course of treatment. Heparin is also used in preventing deep vein thrombosis in patients undergoing surgery. Treatment with heparin and other anticoagulants may, however, have undesirable side effects. Available anticoagulants generally act throughout the body, rather than acting specifically at a clot site. Heparin, for example, may cause heavy bleeding. Furthermore, with a half-life of approximately 80 minutes, heparin is rapidly cleared from the blood, necessitating frequent administrating. Because heparin acts as a cofactor for antithrombin III (AT III), and AT III is rapidly depleted in DIC treatment, it is often difficult to maintain the proper heparin dosage, necessitating continuous monitoring of AT III and heparin levels. Heparin is also ineffective if AT III depletion is extreme. Further, prolonged use of heparin may also increase platelet aggregation and reduce platelet count, and has been implicated in the development of osteoporosis. Indandione derivatives may also have toxic side effects.
Other known anticoagulants comprise Thrombin- and factor Xa inhibitors derived from bloodsucking organisms. Antithrombins, Hirudin, Hirulog and Hirugen are recombinant proteins or peptides derived from the leach Hirudo medicinalis whereas the factor Xa inhibitor antistatin and the derivative rTAP are tick-derived recombinant proteins. Inhibitors of platelet aggregation such as monoclonal antibodies or synthetic peptides, which interfere with platelet receptor GPIIG/IIIa are also effective as anticoagulants.
Bleeding complications are observed as an unwanted major disadvantage of antithrombin, antifactor Xa, as well as antiplatelet reagents. This side effect is strongly decreased or absent with inhibitors of the factor VIIa/TF activity. Such anticoagulants comprise the physiological inhibitor TFPI (tissue factor pathway inhibitor) and modified factor VII (FVIIai), which is factor VII modified in such a way that it is catalytically inactive, but still binds to TF and compete with active factor VIIa.
In addition to the anticoagulants briefly described above, several naturally occurring proteins have been found to have anticoagulant activity. For example, Reutelingsperger (U.S. Pat. No. 4,736,018) isolated anticoagulant proteins from bovine aorta and human umbilical vein arteries. Maki et al. (U.S. Pat. No. 4,732,891) disclose human placenta-derived anticoagulant proteins. In addition, AT III has been proposed as a therapeutic anticoagulant (Schipper et al., Lancet 1 (8069): 854-856 (1978); Jordan, U.S. Pat. No. 4,386,025; Bock et al., U.S. Pat. No. 4,517,294).
Proliferation of smooth muscle cells (SMCs) in the vessel wall is an important event in the formation of vascular lesions in atherosclerosis, after vascular reconstruction or in response to other vascular injury. For example, treatment of atherosclerosis frequently includes the clearing of blocked vessels by angioplasty, endarterectomy or reduction atherectomy, or by bypass grafting, surgical procedures in which atherosclerotic plaques are compressed or removed through catheterization (angioplasty), stripped away from the arterial wall through an incision (endarterectomy) or bypassed with natural or synthetic grafts. These procedures remove the vascular endothelium, disturb the underlying intimal layer, and result in the death of medial SMCs. This injury is followed by medial SMC proliferation and migration into the intima, which characteristically occurs within the first few weeks and up to six months after injury and stops when the overlying endothelial layer is re-established. In humans, these lesions are composed of about 20% cells and 80% extracellular matrix.
In about 30% or more of patients treated by angioplasty, endarterectomy or bypass grafts, thrombosis and/or SMC proliferation in the intima causes re-occlusion of the vessel and consequent failure of the reconstructive surgery. This closure of the vessel subsequent to surgery is known as restenosis.
Modified factor VIIa (FVIIai) has been shown to effectively suppress the restenosis process possibly as a result of a decreased clot formation and thrombin generation initially after treatment of the constricted vessel.
There is still a need in the art for improved compositions having anticoagulant activity which can be administered at relatively low doses and do not produce the undesirable side effects associated with traditional anticoagulant compositions. The present invention fulfils this need by providing anticoagulants that act specifically at sites of injury, and further provides other related advantages such as its effect on the restenosis process. As compared to other anticoagulants with an effect at factor VIIa/TF activity the present invention has the additional advantage that it is a small synthetic molecule suitable for oral administration and for prophylactic treatment of atherosclerotic patients at risk for thrombosis.
It has now surprisingly been found that Zn.sup.2+ -ions exert their inhibitory action in competition with a stimulatory effect of Ca.sup.2+ -ions. It is predicted that Zn.sup.2+ -ions displace Ca.sup.2+ -ions from the calcium binding site believably located in the serine protease domain of FVIIa. The actual site involved is believed to be a Ca site located in a loop of the serine protease domain comprising residues 208-222 of FVIIa (Wildgoose, et al., Biochemistry 32, 114-119(1993); Banner et al. Nature 380:41-46(1996)). This site is located on top of the substrate binding pocket (active site) of the active FVIIa molecule (FIG. 4).
Zinc is very unusual as a ligand for proteases of the chymotrypsin-like serine protease family, to which factor VII belongs. However, zinc is required for catalysis as an obligatory constituent of the active site of another class of proteolytic enzymes, the metalloproteases.
International Patent Publications Nos. WO95/19957, WO92/09563, WO92/09556, WO95/29892, WO95/06031, and WO93/20047 disclose inhibition of several metalloproteinases using hydroxamate-type compounds. These proteases comprise e.g. thermolysin, stromeolysin, collagenase, gelatinase, carboxypeptidases, angiotensin converting enzyme, matrilysin, and enkephalinases. The used hydroxamates provides a bidentate ligand to an essential zinc atom required for catalysis located within the active site and thus blocks proteolysis (Browner et al. Biochemistry 34:6602-6610(1995).
With factor VII it has now surprisingly been found that certain compounds e.g. hydroxamates and hydrazides are capable of acting as powerfull supporters for binding of zinc ions in competition with calcium ions. Thereby specific compounds potentiate zinc inhibition of the activity of the factor VIIa/tissue factor complex. These compounds thus potentiating the inhibition of FVIIa/TF are e.g. dihydroxamates having a spacing from about 0.37 nm to about 0.77 nm, such as 0.57 nm to about 0.67 nm (about 5.7 Angstrom to about 6.7 Angstrom) between their hydroxamate groups.
The mechanism of action for the potentiation of zinc inhibition of FVIIa/TF is thus fundamentally different from the inhibition of metalloproteinases.